Method for the prevention and treatment of bovine mastitis

ABSTRACT

A method for treating or preventing mastitis in cows is disclosed. The method contemplates the intramammary injection of bovine interferon-gamma. Interferon-gamma can be administered prior to infection to effectively suppress the rate, severity, and duration of subsequent bacterial infection, or can be administered subsequent to infection to effectively treat mastitis.

TECHNICAL FIELD

The present invention relates generally to the use of cytokines in theprevention of bacterial infections. More specifically, the instantinvention pertains to the use of interferon-gamma in the prevention andtreatment of bovine mastitis.

BACKGROUND OF THE INVENTION

The mammary gland has a natural ability to prevent bacterial invasion,but various physiological events can inhibit this capability. Forexample, the ability of mammary gland neutrophils to phagocytizemastitis-causing pathogens and suppress bacterial multiplication iscritical to, the outcome of intramammary infection (Paape et al., 1979).Although both neutrophils and macrophages predominate in mammarysecretions and tissues (Sordillo et al., 1987; Sordillo and Nickerson,1988), evidence suggests that the antibacterial activities of thesecells are reduced in the presence of mammary secretions and compromisedduring physiological transitions of the gland (Paape et al., 1981;Nagahata et al., 1988; Sordillo and Nickerson, 1988). Consequently, thebovine mammary gland is highly susceptible to mastitis immediatelyfollowing the cessation of lactation and during the periparturientperiod (Nickerson, 1989). Incidence of clinical mastitis is highestduring early lactation often resulting from new intramammary infectionsobtained during the nonlactating period. Increased susceptibility duringthese times is most likely due to a combination of increased exposure ofteat ends to mastitis-causing pathogens and diminished host defensemechanisms, as described above, during functional transitions of themammary gland.

Mastitis during the periparturient and early lactating periods is causedby a multitude of bacteria. Common etiological agents includeEscherichia coli and Staphylococcus sp. Coliform infection is rare inmiddle and late lactation, but is most severe during the first few weeksof lactation where it is a major cause of acute toxic mastitis.Disparities in establishment and severity of, coliform mastitis withrespect to stage of lactation have been explained by the rate of growthof the organism within the gland, the elaboration and absorption oftoxins, and the varying susceptibility of the host during these stages.A rapid and intense inflammatory response in lactating tissue has beenobserved following experimental challenge with E. coli (Hill, 1981). Inmost cases, the bacteria were eliminated rapidly without causing damageto secretory parenchymal tissue. In contrast, quarters infected with E.coli during the immediate postpartum period had minimal neutrophilinflux, which probably allows the unrestricted growth of the organism.The delayed diapedesis of neutrophils and slow inflammatory responsewithin the gland may be a result of decreased sensitivity of alveolarand ductular epithelium to endotoxins during functional transitions ofthe gland.

Interferons (IFN) are a family of closely related proteins of threemajor types (Lawman et al., 1989). Interferons-alpha and -beta areproduced by a variety of cell types in response to several inducersincluding viral infections, bacterial products and tumor cells.Interferon-gamma is predominantly produced by antigen- ormitogen-stimulated T-lymphocytes. In addition to the antiviral andantiproliferative activities, all classes of IFN are known to exhibitmany immunomodulatory properties (Lawman et al., 1989).

IFN-gamma has been shown to be a potent immunomodulator and appears toenhance natural killer cell activity, antibody-dependent cellularcytotoxicity, and cytotoxic T-lymphocyte activity (Lawman et al., 1989).Interferon-gamma also enhances macrophage-mediated cytotoxicity againsttumor cells, induces membrane-bound Fc receptors for IgG on macrophages,and stimulates the synthesis and release of reactive oxygen metabolitesfrom both macrophages and neutrophils (Bielefeldt Ohmann and Babiuk,1986; Trinchieri and Perussia, 1985).

The efficacy of interferons in the treatment of mastitis has notheretofore been studied to applicants' knowledge.

SUMMARY OF THE INVENTION

The present invention is based on the surprising discovery thatinterferons can protect dairy cattle against coliform mastitis.

In one embodiment the present invention is directed to a method oftreating or preventing mastitis in a mammal comprising administering tosaid mammal a therapeutically effective amount of interferon.

In another embodiment, the instant invention is directed to a method fortreating or preventing coliform mastitis in a cow comprisingadministering to said cow a therapeutically effective amount of bovineinterferon-gamma. The administration can be done either before or afterinfection.

These and other embodiments of the present invention will readily occurto those of ordinary skill in the art in view of the disclosure herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the effect of increasing IFN-gamma doses on bacterialphagocytosis of bovine mammary neutrophils pretreated with eitherperiparturient mammary secretions (milk) or Hank's balanced salinesolution (HBSS). Data are expressed as colony-forming units (CFU) ofStaphylococcus aureus recovered from mammary neutrophils at t=0. Barsbetween IFN-gamma treatments with different letters (a,b) aresignificantly different (P<0.05).

FIG. 2 shows the effect of increasing IFN-gamma doses on bactericidalactivity of bovine mammary neutrophils pretreated with eitherperiparturient mammary secretions (milk) or Hank's balanced salinesolution (HBSS). Bars between IFN-gamma treatments with differentletters (a,b) are significantly different (P<0.05).

FIG. 3 demonstrates the effect of increasing IFN-gamma doses on theproduction or reactive oxygen species (ROS) by bovine mammaryneutrophils pretreated with either periparturient mammary secretions(milk) or Hank's balanced saline solution (HBSS). Data are expressed astotal area under the curve.

FIG. 4 shows the effect of recombinant bovine IFN-gamma on the rate andduration of experimental E. coli-induced bovine mastitis during theimmediate portpartum period.

FIG. 5 depicts the effect of IFN-gamma treatment on the rate andduration of experimental E. coli-induced bovine mastitis during theprepartum period.

DETAILED DESCRIPTION

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of immunology, protein chemistry,biochemistry and molecular biology which are within the skill of theart. Such techniques are explained fully in the literature. See, e.g.,Handbook of Experimental Immunology, Vols. I-IV (D. M. Weir and C. C.Blackwell eds., 1986, Blackwell Scientific Publications), and the seriesMethods in Enzymology (S. Colowick and N. Kaplan eds., Academic Press,Inc.)

The present invention is directed to the treatment or prevention ofmastitis in mammals by the administration of one or more interferons tothe subject mammal. A preferred embodiment of the invention is thetreatment or prevention of coliform mastitis in cattle using bovineinterferon (BoIFN)-gamma.

The present invention contemplates employing any form of human or BoIFN,either alone or in combination. Thus, the present invention encompassesusing native forms of the interferons. Particularly surprising, however,is that recombinant forms of the interferons have sufficient biologicalactivity to provide for effective treatment or prevention of mastitis.Since the production of recombinant interferon has substantialadvantages relative to the purification of native polypeptides,recombinant interferons are a preferred embodiment. It is alsocontemplated that synthetic forms of human or BoIFN, and muteins orfragments of BoIFN, exhibiting effective biological activity, are alsowithin the scope of the invention. A mutein of human or BoIFN is apolypeptide substantially homologous to the native protein, and morehomologous to the bovine or human form than to IFNs from other species.A fragment of human or BoIFN is a polypeptide that is homologous to aregion of sufficient length in the protein such that the sequence isunique to human or BoIFN.

The production of recombinant animal interferons, including alpha(leukocyte) and gamma (immune), is known in the art. See, e.g., EPO Pub.No. 088,622, by D. Capon & D. Goeddel. Furthermore, recombinantBoIFN-gamma is available commercially from Ciba-Geigy, Ltd., Basel,Switzerland.

As indicated, the present invention is concerned with treating orpreventing mastitis. By "treating" is meant curing or ameliorating ananimal that has contracted mastitis. "Preventing" mastitis meanspreventing the occurrence of the infection, or tempering the severity ofthe infection if it is later contracted.

Typically, the interferons of the present invention are administered byintramammary injection; however, effective dosages may be administeredvia intramuscular, subcutaneous, or intravenous injection. When preparedas injectables, the interferons are generally administered using apharmaceutically acceptable vehicle or excipient. Suitable vehicles are,for example, water, saline, dextrose, glycerol, ethanol, or the like,and combinations thereof. In addition, if desired, the vehicle maycontain auxiliary substances such as wetting or emulsifying agents or pHbuffering agents. The active ingredient will typically range from about1% to about 95% (w/w) of the composition administered, or even higher orlower if appropriate.

A "therapeutically effective amount" of interferon is a dose sufficientto either prevent or treat mastitis in a subject to which the interferonis administered. The dosages of the interferons which can treat orprevent mastitis can be determined in view of this disclosure by one ofordinary skill in the art by running routine trials with appropriatecontrols. Comparison of the appropriate treatment groups to the controlswill indicate whether a particular dosage is effective in preventing ortreating a disease used in a controlled challenge. In general, effectivedosage will vary depending on the mode of administration. It has beenfound that in the case of an intramammary injection using recombinantBoINF-gamma, administration of 10⁵ U per quarter is sufficient to retardE. coli mastitis.

If administered intramuscularly, subcutaneously, or intravenously,effective dosages will depend on the weight of the animal and willtypically run in the range of from about 0.1 ug/kg to about 40 ug/kg.More typically, the dosage will be at least about 1 ug/kg, but less than10 ug/kg. On an absolute basis, single-dose formulations will containfrom about 10 ug to about 1,000 ug. More typically, preferredsingle-dose formulations will be at least about 30 ug, 100 ug or even200 ug, but usually no more than about 600 ug or 700 ug. Effectivedosages are believed to fall within a relatively large range.

Beyond dosage, an effective administration of interferons according tothe present invention will in part depend on the number and timing ofthe dosages. For example, multiple administrations of a dosage may begiven to an animal, typically at least about 24 hours apart. In somecircumstances it may be desirable to administer the interferon at leasttwice to the animal, or at least three times. It may even be desirableto administer even more dosages to the animal, such as six, seven,eight, or even nine over an equal number of days or longer. Again, it isbelieved that the precise combination of dosage and timing will besubject to a wide range of variation and that numerous combinationseffective in treating or preventing a disease can be readily establishedby those of ordinary skill in the art in view of the present disclosure.

The interferons of the subject invention can be administered prior toinfection, and thus serve as a prophylactic or can be given after thesubject has shown signs of infection.

Described below are several examples illustrating the practice of thepresent invention. These examples are provided for illustrative purposesonly, and are not intended to limit the scope of the appended claims.

EXPERIMENTAL EXAMPLE 1

Preliminary experiments were performed to determine the effect ofrecombinant BoIFN on mammary neutrophil activity during theperiparturient period.

Mammary Neutrophil Isolation

Neutrophils were isolated from the mammary gland of a heifer afterintramammary injection of 5 ug of lipopolysaccharide (LPS) in 5 ml ofHBSS. At 15 h after LPS administration, 30 ml of HBSS were injected intothe LPS-treated quarter and mammary secretions were collected into asterile polypropylene tube. Mammary neutrophils were purified byFicoll-Hypaque density centrifugation. After 2 washings, the cells wereresuspended in HBSS containing 5% fetal bovine serum (FBS) and filteredthrough a 30 um Nitex filter (Tetko Co., Elmsford, N.Y.). Separation ofmammary leukocytes via this technique consistently resulted in a morethan 96% neutrophil population. The percentage of viable cells wasdetermined by hemacytometer count using trypan blue exclusion. Mammaryneutrophils were cytocentrifuged onto poly-L-lysine-coated slides andstained with Wright,s stain for differential cell counts.

Pretreatment and Stimulation of Mammary Neutrophil

Mammary secretion samples from individual quarters (n=8) were collectedfrom 2 cows at approximately 14 and 7 days prior (±2 days) to expectedparturition (C-14 and C-7) and at parturition (C-0). Aseptic foremilksamples were collected in duplicate and used to determine infectionstatus of each quarter. Bacteriologic evaluation of foremilk samplesrevealed that all quarters (n=8) were free of intramammary infectionduring the entire sampling period. Secretions were centrifuged for 30min (3,000 g at 5° C.) to remove fat and cellular debris (skim milk).

Skim milk preparations (1 ml) from each sampling period were mixed with1 ml of mammary neutrophils (8×10⁶ cells) and allowed to incubate for 1h at room temperature. Control cells were incubated in HBSS. The mixturewas washed in HBSS and separated into four 1-ml volumes containing 2×10⁶cells each. For each sampling period, skim milk-treated neutrophils weresubsequently incubated with 10 U, 100 U and 1000 U of recombinantBoIFN-gamma (Ciba-Geigy Ltd., Basel, Switzerland) or HBSS for 2 h atroom temperature. The cells were washed and resuspended in HBSS with 5%FBS to a final concentration of 1×10⁶ cells/ml.

Phagocytic and Bacteriocidal Assay

Mammary neutrophil phagocytic and bactericidal capacities were evaluatedusing Staphylococcus aureus isolated from a clinical case of bovinemastitis. Bacteria in log phase growth were opsonized with bovine serawith an agglutination titer of 1/16 against S. aureus.

Opsonized S. aureus cells (1×10⁶ colony-forming cells (CFU) in 1 ml)were mixed with 1 ml of treated neutrophils (1×10⁶) and incubated (10min, 37° C.) in a shaking water bath. After initial incubation, themixtures were washed, resuspended in HBSS containing lysostaphin (SigmaChemical Co., St. Louis, Mo.), and incubated for 30 min to removeextracellular bacteria. Cells were washed and divided into 3 aliquots:t=0 min, t=30 min, and t=60 min. Following the second incubation, PMNwere washed, resuspended in 1 ml HBSS, and subjected to sonication.

The number of viable bacteria for each time period was estimated by astandard plate count method. The mean CFU of bacteria at t=0 was used asan index of phagocytosis. Bactericidal capacity (percent killing) wascomputed from: ##EQU1##

Chemiluminescence

Luminol-enhanced chemiluminescence was measured with a Packard Picoliteluminometer (Packard Instrument Co., Downers Grove, Ill.). Treatedneutrophils (1×10⁶ cells/100 ul) were added to vials containing 15 ulluminol, 200 ul of opsonized zymosan (10 mg/ml), and 1,685 ul HBSS.Vials were counted for 10 s at 7 min intervals for a total of 210 min.All assays were performed in duplicate. For each quarter, mammarysecretion from all sampling periods and the appropriate controls wereassayed on the same day.

Transmission Electron Microscopy

Mixtures containing skim milk-treated neutrophils, recombinantBoIFN-gamma, and S. aureus from one quarter were fixed in 2.5%glutaraldehyde in 0.1M cacodylate buffer for 2 h and postfixed in 0.1Mcacodylate-buffered osmium tetroxide for 1.5 h. Following dehydration ina graded series of ethanol washes, cell pellets were embedded in epoxyresins. Ultrathin sections (60 nm) for electron microscopy were stainedwith 5% uranyl acetate in 50% methanol for 20 min followed by 0.4% leadcitrate for 10 min. Sections were examined using a Philips 300transmission electron microscope (Philips Export, Eindhoven,Netherlands) operating at 60 kV.

Statistical Analysis

Data were analyzed by least-squares analysis of variance using thegeneral linear model procedure to determine effects of mammary secretionand increasing doses of recombinant BoIFN-gamma on mammary glandneutrophil function. Statistical analysis included effect of cow,mammary secretion, recombinant BoIFN-gamma, and interaction of mammarysecretions with recombinant BoIFN-gamma. Preplanned comparisons of leastsquare means from the overall model were made by pairwise t-test.

Results

The effects of periparturient mammary secretion of neutrophil functionare shown in Table 1. Mammary neutrophils treated with secretionsobtained during the last 2 weeks of gestation had significantly lowerphagocytic and bactericidal activity when compared with control cellsincubated in HBSS. Cells incubated with skim milk preparations also hadsignificantly less production of reactive oxygen species (ROS) whencompared with control neutrophils incubated in HBSS. Secretions obtainedat C-7 and C-0 tended to inhibit neutrophil function more thansecretions obtained at C-14.

Ultrastructural examination of skim-milk-treated neutrophils frequentlyrevealed phagocytic vacuoles containing casein-like micelles and debrisin addition to ingested bacteria. In contrast, control neutrophilsincubated in HBSS primarily had internalized bacteria with very littleingested debris.

Treatment with recombinant BoIFN-gamma significantly increased thephagocytic and bactericidal capacity of neutrophils for S. aureus (FIGS.1 and 2) for both skim milk-treated and control cells. However, skimmilk-treated neutrophils consistently showed lower phagocytic andbactericidal capabilities when compared with cells incubated in HBSS.There were no significant effects of recombinant BoIFN-gamma onproduction of ROS by mammary neutrophils although values tended to beslightly higher following recombinant BoIFN-gamma treatment (FIG. 3).

There were no apparent dose-dependent responses of skim milk-treatedneutrophils to increasing recombinant BoIFN-gamma concentrations forbacterial phagocytosis, bactericidal activity, of ROS generation. Incontrast, HBSS-treated neutrophils showed a trend of enhanced responsewith increasing doses of recombinant BoIFN-gamma for all parametersmeasured (FIGS. 1-3).

                  TABLE 1                                                         ______________________________________                                        Effects of periparturient mammary secretions on                               polymorphonuclear leukocyte function in vitro                                                    Bacterial   ROS                                            Secretion                                                                            Phagocytosis                                                                              Activity    Production.sup.2                               Sample (CFU/ml).sup.-3                                                                           (% kill)    (1 × 10.sup.7)                           ______________________________________                                        HBSS   1.5.sup.a ± 0.14                                                                       81.9.sup.a ± 3.5                                                                       247.6.sup.a ± 19.2                          C-0.sup.1                                                                            1.0.sup.b ± 0.10                                                                       61.6.sup.b,c ± 2.5                                                                     172.1.sup.c ± 13.6                          C-7    1.0.sup.b ± 0.10                                                                       57.9.sup.c ± 2.5                                                                       181.9.sup.b,c ± 13.6                        C-14   1.3.sup.a,b ± 0.10                                                                     69.1.sup.b ± 2.5                                                                       217.3.sup.a,b ± 13.6                        ______________________________________                                         .sup.a,b,c Means within each parameter with different superscripts differ     (p < 0.05).                                                                   .sup.1 Days preceding calving (C).                                            .sup.2 Data expressed as total area under the curve over a 203 min period                                                                              

Thus, treatment of mammary neutrophils with recombinant BoIFN-gammareversed the suppressive effects of mammary secretion, resulting inhigher chemiluminescent activity and significantly more bacterialphagocytosis when compared with untreated controls. Results of thisstudy suggested that recombinant BoIFN-gamma may have someimmunomodulatory value for the prevention and treatment of bovinemastitis during the periparturient period.

To test this hypothesis, the following experiment was performed.

EXAMPLE 2

The influence of recombinant BoIFN-gamma on the establishment, severity,and duration of experimentally induced E. coli mastitis in dairy cattlewas tested using the following method.

Experimental Animals

Eight Holstein-Frisian dairy cows were used in this study. These cowswere purchased from local dairy producers. Animals arrived at thetesting facility during their last trimester of pregnancy and wereallowed to acclimate at least one month prior to experimentalmanipulation. Duplicate quarter foremilk samples were collectedaseptically, immediately following arrival to determine infection statusof each quarter. Bacteriologically positive quarters were treated withappropriate lactating intramammary antibiotic preparations based onsensitivity tests. The withdrawing time for antibiotic preparations was72 hours. Animals were resampled approximately one week prior toexperimental manipulation to ensure all quarters were free ofintramammary infection. Only cows with healthy appearances and normalrectal temperatures, heart rates, and respiration were used in thistrial.

Experimental Design

Cows were randomly assigned to one of four treatment groups as outlinedin the following table:

                  TABLE 2                                                         ______________________________________                                        Treatment groups                                                              Group # Lactation Stage                                                                              Treatment*                                             ______________________________________                                        1        4 days prepartum                                                                            10.sup.5 U IFN/qtr at -24 hour                         2        4 days prepartum                                                                            control, no IFN treatment                              3       ≦7 days postpartum                                                                    10.sup.5 U IFN/qtr at -24 hour                         4       ≦7 days postpartum                                                                    control, no IFN treatment                              ______________________________________                                         *Treated cows were intramammarily infused into all four quarters (qtr)        with 10.sup.5 U recombinant BoIFNgamma at 24 hours prior to experimental      challenge. All animals were challenged with approximately 50 CFU of E.        coli into each quarter.                                                  

Mammary quarter secretion samples, sera, and rectal temperatures werecollected from each group as outlined in the following table:

                                      TABLE 3                                     __________________________________________________________________________    Sample schedule (hours)                                                       Group                                                                             Time Post-infusion                                                                           Time Post-challenge                                        #   0  4  6  8  12 0  4  6  8  12 24 36 48 60                                 __________________________________________________________________________    1   x     x     x  x     x     x  x  x  x  x                                  2                  x     x     x  x  x  x  x                                  3   x  x     x  x  x  x     x  x  x  x  x  x                                  4                  x  x     x  x  x  x  x  x                                  __________________________________________________________________________

In groups 1 and 2, all samples were collected again at calving and at 3,7, and 14 days post-calving. In groups 3 and 4, all samples werecollected again at 7 and 14 days post-challenge. During all samplingperiods, clinical status of each quarter was recorded. Mammary secretionsamples were used to determine infection status and to quantitateconcentrations of lactose, fat, total protein, serum albumin, andlactoferrin. Total somatic cell counts, pH, and cytokine levels werealso determined. Blood samples were used to determine total anddifferential cell counts and to quantitate concentrations of totalprotein, fibrinogen, zinc, iron, and copper.

Quarter milk production was recorded at each milking (8:30 a.m. and 8:30p.m.). Mammary gland preparation for milking consisted of a prewash witha disinfectant iodine solution and drying with individual paper towels.Prior to each milking, a strip cup was used to observe clinical signs ofmastitis in the first few streams of foremilk. Immediately followingmilking, teats were dipped with a 1% iodophor solution.

At 14 days post-challenge, the cows were slaughtered and approximately 1cm³ of tissue was obtained from each quarter. Tissue specimens wereprepared for both light and electron microscopy.

IFN-gamma infusion

Mammary glands were washed with a disinfectant iodine solution andthoroughly dried with a single service paper towel. A few streams offoremilk were stripped from each quarter. Teat ends were scrubbed withswabs containing 70% isopropyl alcohol. Infusions of IFN-gamma werediluted in 10 ml of sterile saline to a final treatment concentration of10⁵ U recombinant BoIFN-gamma per quarter. A sterile 10 ml syringefitted with an infusion canula was used to deliver the IFN-gammasolution into the streak canal of each quarter. The IFN-gamma infusionwas massaged up into the gland cistern and teats were dipped with adisinfectant iodine solution.

Challenge inoculum

E. coli was isolated from an acute case of bovine mastitis from theUniversity of Saskatchewan Research Herd. The gram-negative isolateswere typed using the API-2pE typing system. Stock cultures of E.coli-V374 were stored at -70° C. in skim milk until needed.

The bacterial challenge was prepared by streaking the stock culture ofE. coli-V374 onto Esculin Blood Agar plates containing 5% whole blood.After 48 hours incubation at 37° C., a single colony was used toinoculate 100 ml of Ultra High Temperature (UHT) pasteurized milk andincubated for 12 hours at 37° C. The 12 hour culture was mixed well anda 100 ul aliquot removed to inoculate a second 100 ml of UHT milk. Aftera 6 hour incubation at 37° C., the culture was serially diluted in10-fold increments using sterile saline. The colony-forming units(CFU)/ml of each dilution was determined by plate pouring 100 ul of thebacterial suspension onto McConkey agar plates. The dilution containing50 CFU of E. coli/ml of saline was selected from each trial. Organismswere refrigerated during the time interval between inoculum preparationand actual infusion (approximately 24 hours). Inocula were platedpost-challenge to confirm viability counts.

Results

The actual bacterial counts used to challenge each experimental animalwere as follows:

    ______________________________________                                                 CFU/         IFN-       CFU/                                         CONTROLS QUARTER      TREATED    QUARTER                                      ______________________________________                                        088      50           005        50                                           .sup. 006.sup.a                                                                        75           .sup. 007.sup.a                                                                          75                                           .sup. 033.sup.b                                                                        30           .sup. 013.sup.b                                                                          30                                           048      50           040        50                                           ______________________________________                                    

Animals between treatment groups with the same superscript (a,b) werechallenged on the same day with the same bacterial suspension. Theefficiency of establishing E. coli mastitis with this model system tomimic acute coliform mastitis as it occurs in the field is summarized inTable 4.

                                      TABLE 4                                     __________________________________________________________________________    Rate of experimental intramammary infection in dairy cattle with an E.        coli mastitis model                                                           Average CFU                                                                              # Eligible Quarters.sup.1                                                                  # Infected Quarters                                                                        % Success                                __________________________________________________________________________    51         15           13           86                                       __________________________________________________________________________     .sup.1 Quarters which were not pretreated with recombinant BoIFNgamma         prior to experimental challenge with E. coli Strain V374                 

The clinical status of all quarters was recorded immediately prior toexperimental challenge and for 26 consecutive observations made at 12hour intervals. The clinical scores were recorded on a scale from 1 to 5where: 1 is normal milk with no quarter swelling; 2 is questionable milkbut no quarter swelling; 3 is obvious abnormal milk but no quarterswelling; 4 is abnormal milk and swollen and/or tender quarter; and 5 isacute mastitis with systemic involvement.

The results of this study can be seen in Tables 5 and 6 and FIGS. 4 and5.

                  TABLE 5                                                         ______________________________________                                        Effects of recombinant BoIFN-gamma on the severity                            of experimental E. coli mastitis                                                                 Average    Average                                         Treatment #        % Days     Clinical                                                                             %                                        Group     Quarters Clinical.sup.1                                                                           Scores.sup.2                                                                         Mortality                                ______________________________________                                        Prepartum                                                                     IFN       8        17.79      1.32    0                                       CONTROL   8        90.39      3.09   50                                       Postpartum                                                                    IFN       8        15.39      1.33    0                                       CONTROL   7        60.90      1.93   50                                       Total                                                                         IFN       16       16.59      1.33    0                                       CONTROL   15       75.64      2.51   50                                       ______________________________________                                         .sup.1 Average % days that quarters showed clinical signs of mastitis         (i.e., clinical score of 2 or above).                                         .sup.2 Average clinical score calculated from the 26 consecutive              observations following challenge.                                        

Those cows treated with interferon had significantly lower clinicalscores, especially in the prepartum period. Furthermore, IFN-treatedcows had a 0% mortality rate attributed to mastitis, whereas the controlgroup had a 50% mortality rate (Table 5). IFN-gamma effectively reducedthe percentage of infected quarters (Table 6 and FIG. 4), and reducedthe rate and duration of experimental E. coli mastitis during theprepartum period (FIG. 5).

                  TABLE 6                                                         ______________________________________                                        Efficacy of recombinant BoIFN-gamma against                                   experimental E. coli mastitis in dairy cattle                                           Quarter: Quarter:  Quarter:                                                   # at     # infected                                                                              % infected                                       Treatment beginning                                                                              at end of at end of                                                                             %                                        Groups    of trial trial     trial   Reduction                                ______________________________________                                        Prepartum                                                                     IFN       8        2         25      71.4                                     CONTROL   8        7         87.5                                             Postpartum                                                                    IFN       8        1         12.5    85.4                                     CONTROL   7        6         85.7                                             Total                                                                         IFN       16       3         23.1    73.4                                     CONTROL   15       13        86.7                                             ______________________________________                                    

Thus, an effective treatment for mastitis has been demonstrated.Although preferred embodiments of the subject invention have beendescribed in some detail, it is understood that obvious variations canbe made without departing from the spirit and the scope of the inventionas defined by the appended claims.

REFERENCES

Bielefeldt Ohmann, H., and I. A. Babiuk. 1986. Alteration of someleukocyte functions following in vivo and in vitro exposure torecombinant alpha and gamma interferon. J. Interferon Res., 6:123.

Hartmann, P. E. 1973. Changes in the composition and yield of mammarysecretion of cows during the initiation of lactation. J. Endocrinol.59:231.

Hill, A. W. 1981. Factors Influencing the Outcome of Escherichia coliMastitis in the Dairy Cow. Res. Vet. Sci. 31:107.

Lappegard, K. T., H. B. Benestad, and H. Rollog. 1988. Interferonsaffect oxygen metabolism in human neutrophil granulocytes. J. InterferonRes. 8:665.

Lawman, M. J. P., M. Campos, H. Bielefeldt Ohmann, and L. A. Babiuk.1989. Recombinant cytokines and their potential therapeutic value inveterinary medicine. In: Comprehensive Biotech. Pergamon Press, London.In press.

Miller, R. H., U. Emanuelson, and E. Perrson. 1983. Relationships ofmilk somatic cell counts to daily milk yield and composition. ActaAgric. Scand. 33:209.

Newbold, F. H. S., and F. K. Neave. 1965. The recovery of small numbersof Staphylococcus aureus infused into the bovine teat cistern. J. DairyRes. 32:157.

Nagahata, H., S. Makino, H. Takahashi, and H. Noda. 1988. Assessment ofneutrophil function in the dairy cow during the perinatal period. J.Vet. Med. 35:747-751.

Nickerson, S. C. 1989. Immunological aspects of mammary involution. J.Dairy Sci. In press.

Paape, M. J., and N. J. Carlett. 1984. Intensification of milk somaticcell response to intramammary device. Am. J. Vet. Res. 45:1572.

Paape, M. J., W. P. Wergin, A. J. Guidry, and W. D. Schultze. 1981.Phagocytic defense of the ruminant mammary gland. Adv. Exp. Med. Biol.137:555.

Sordillo, L. M., S. C. Nickerson, and R. M. Akers. 1989. Pathology ofStaphylococcus aureus mastitis during lactogenesis: relationship withbovine mammary structure and function. J. Dairy Sci. 72:228-240.

Sordillo, L. M., S. C. Nickerson, R. M. Akers, and S. P. Oliver. 1987.Secretion composition during bovine mammary involution and therelationship with mastitis. Int. J. Biochem. 19:1165.

Sordillo, L. M., and S. C. Nickerson. 1988. Morphological changes in thebovine mammary gland during involution and lactogenesis. Am. J. Vet.Res. 49:1112.

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We claim:
 1. A method of treating or preventing mastitis in a mammal comprising administering to said mammal by intramammary injection a therapeutically effective amount of interferon.
 2. The method of claim 1 wherein said interferon is interferon-gamma.
 3. The method of claim 2 wherein said mammal is a cow.
 4. The method of claim 3 wherein said interferon-gamma is recombinant bovine interferon-gamma.
 5. A method for treating or preventing coliform mastitis in a cow comprising administering to said cow by intramammary injection a therapeutically effective amount of bovine interferon-gamma.
 6. The method of claim 5 wherein said injection is given during the prepartum period.
 7. The method of claim 5 wherein said injection is given during the postpartum period.
 8. The method of claim 5 wherein said interferon-gamma is administered prior to the onset of the infection.
 9. The method of claim 5 wherein said interferon-gamma is administered after the onset of infection. 